Antigen receptors with diverse binding activities are the hallmark of B and T cells of the adaptive immune system in jawed vertebrates and are generated by genomic rearrangement of variable (V), diversity (D), and joining (J) gene segments separated by highly variable junction regions (Schatz (2004) Semin. Immunol. 16, 245-256). Initial calculations have been made of the combinatorial and junctional possibilities that contribute to the human immune receptor repertoire, and it is estimated that the number of possibilities may greatly exceed the total number of peripheral T or B cells in an individual (Davis and Bjorkman (1988) Nature 334, 395-402).
For example, one study in which small subsets of rearranged T cell receptor (TCR) subunit genes were extensively sequenced with a few segment-specific primers yielded extrapolations for the full TCR repertoire corresponding to 2.5×107 distinct TCRα-TCRβ pairs in the peripheral blood of an individual (Arstila et al. (1999) Science 286, 958-961). Extensive repertoire analyses for the human B cell compartment have been more limited, although small-scale studies and focused analysis of immunoglobulin (Ig) class subsets, such as IgE, have been performed (Brezinschek et al. (1995) J. Immunol. 155, 190-202, Lim et al. (2007) J. Allergy Clin. Immunol. 120, 696-706). Advanced sequencing methods have recently been used to analyze B cell receptor diversity in the relatively simple model immune system in zebrafish (Weinstein et al. (2009) Science 324, 807-810).
Against a background of continually generated novel DNA sequences, expanded clones of lymphocytes with useful antigen specificities persist over time to enable rapid responses to antigens previously detected by the immune system. Systematic means for detection of such expanded clones in human beings would provide significant opportunities for specific analysis and tracking, including measurement of clonal population sizes, anatomic distributions, and changes in response to immunological events.
In contrast to healthy immune systems, malignancies of B or T cell origin typically express a single dominant clonal Ig or TCR receptor. A variety of assays have been used to detect the presence of B cell clonality for diagnosis of lymphomas and leukemias, including analysis of Ig light chain gene restriction and Southern blotting or sizing of polymerase chain reaction (PCR) products from rearranged Ig or TCR loci (Rezuke et al. (1997) Clin. Chem. 43, 1814-1823; Arber (2000) J. Mol. Diagn. 2, 178-190). Although adequate for many applications, these strategies make limited use of the high information content inherent in rearranged immune receptor gene sequences and can give indeterminate results.
A recent study using deep sequencing of clonal IgH (Ig heavy chain) receptor genes in chronic lymphocytic leukemia revealed unexpected intraclonal heterogeneity in a subset of cases, showing that previous approaches have not captured the fundamental features of leukemic cell populations (Campbell et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 13081-13086). Detection of more subtle clonal populations (for example, to follow the response of lymphomas or leukemias to treatment) now relies on time- and labor-intensive multiparameter flow cytometry or custom-designed patient- and clone-specific realtime PCR assays (Sayala et al. (2007) Best Pract. Res. Clin. Haematol. 20, 499-512; Ladetto et al. (2000) Biol. Blood Marrow Transplant. 6, 241-253). Early diagnostic screening approaches may benefit from generalized and more efficient clonal detection. Indeed, a recent population-based epidemiological study showed that small amplified B cell populations can be seen in almost all individuals who go on to develop chronic lymphocytic leukemia, further underscoring the importance of assessing lymphocyte clonality in human specimens.
Detection and analysis of clonality is also of fundamental interest in characterizing and tracking normal and pathogenic immune reactions. For protective and healthy humoral immune responses, high-resolution analysis of immune receptor clonality and evolution offers the potential for definitive detection and monitoring of effective immune responses to vaccination and specific infections, whereas for some autoimmune disorders this type of analysis may facilitate diagnosis, long-term therapeutic monitoring strategies, and, eventually, specific interventions.